The applicant proposes to use an in vitro procedure that detects drug-resistant genetic variants of HIV reverse transcriptase (RT) expressed in bacteria. Employing a recently developed, novel in situ colony screening assay for HIV RT, substrate analog inhibitor-resistant HIV RT enzymes will be isolated. Cross-resistance of such mutant RTs to other drugs will be determined to help plan combination therapy. Selected mutations will be rebuilt into an infectious molecular clone of HIV and will be tested for replication competence. This will be done in an attempt to test the in vivo relevance of the mutant RTs with decreased sensitivity to substrate analog inhibitors obtained by in situ colony screening. Furthermore, the investigator proposes to characterize mutant RTs with respect to the site of mutation, rates of misincorporation and other aspects of DNA polymerization. To gain further insights into substrate-binding sites, residues in and around the mutations will be further examined by specific mutagenesis. Resistance detected biochemically may or may not reflect resistance during replication in vivo. To ascertain the ability to anticipate mutations that arise in vivo by utilizing this simple in vitro approach, viruses will be isolated from patients who are under therapy in AIDS clinical trials and their RT sequences will be tested for the presence of mutations that lead to resistance.